Infections caused by Mycobacterium avium complex are common in AIDS patients, and in patients with chronic underlying lung disease, such as emphysema and bronchiectasy. Despite of the success of anti-HIV-1 therapy in reducing the cases of M.avium disease, recent published work has demonstrated that in many areas M.avium is M.avium is an environmental bacteria for which increasing incidence of disease in humans is predictable by the increased percent of the population with predisposing conditions The ability to survive in different environments in the host requires tight gene regulation. Because M.avium is resistant to most of the antibiotic markers and is difficult to transform by external genetic material, it was necessary to create specific systems to study pathogenesis. During the past 3.5 years of this grant we have developed or adapted molecular systems, such as a transposon mutagenesis, signature tagged mutagenesis (STM), and GFP promoter fusion library, that allowed us to begin dissecting the complex aspects of M.avium interaction with the host. Several virulence genes were identified that allow the bacterium to enter intestinal epithelial cells, and to survive in macrophages. In addition, the great majority of the genes identified in vitro are also associated with virulence in vivo. Our hypothesis is that M.avium has specific strategies to subvert the host cells. We propose to continue this work by: A- Investigating how M.avium virulence-related genes are involved in the mechanism of invasion of intestinal mucosal cells. Our studies thus far have determined that bacterial entry is associated with the activation of small GTPases Rho A, and Cdc 42, and phosphorylation of N-WASP. We now detail experiments to further dissect the host cells pathways needed for bacterial uptake, based on the hypothesis that two pathways are used to enter epithelial cells. B- Analyzing the function of virulence determinants that are involved in the ability to survive and replicate in macrophages. We have developed a screening for the isolation of transposon-mutagenized M.avium bacteria that does not inhibit phagosome-lysosome fusion, and fails to suppress vacuole acidification. An initial screen of 3000 mutants resulted in the identification of a number of virulent determinants, such as PPE genes, polyketide synthases, MmpL proteins, ABC transporters, and several genes of unknown function.